Title

Author

Degree Type

Dissertation

Date of Award

2013

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Basil J. Nikolau

Abstract

Wax esters occur widely among bacteria, plants and mammals. Due to their special properties, they have many applications such as lubricants, cosmetics, pharmaceutical products, ink, polishes and candles. They are also considered as potential biofuels and biolubricants in the future. Wax ester producing enzymes synthesize wax esters from alcohols and fatty acyl coenzyme A thioesters. There are three gene families encoding enzymes capable of synthesizing wax esters. Two of them exist in plans; the jojoba-like wax synthase (WS), and the bifunctional wax synthase/diacylglycerol acyl transferase (WS/DGAT). We investigated the phylogenetic relationships among and between WS and WS/DGAT, based upon primary sequence homology of the encoded proteins. Nine candidate genes were chosen for experimental characterization in yeast and Arabidopsis seed.

In yeast heterologous expression system, three of the expressed gene products were detected immunologically, Arabidopsis WS At5g55340, a maize WS and a moss WS. Moreover, by feeding these transgenic yeast strains with potential fatty acid and fatty alcohol substrates, we demonstrated that At5g55340 and the maize WS were expressed in a functional state, and could direct the synthesis of novel ester lipids. Based upon the ability to form different esters from different precursors, we were able to deduce the substrate preferences of these two enzymes. These characterizations indicate that At5g55340-encoded WS has a rather narrow substrate preference, producing esters with linear fatty acids and fatty alcohols of about equal chain length. In contrast the maize WS has a wider substrate preference, producing similar linear esters as At5g55340, but also benzyl and ethyl esters.

These candidate genes were also expressed in seeds of Arabidopsis Col-0. These transgenic events were coupled with the co-expression of the jojoba 3-ketoacyl-CoA synthase (KCS) and the jojoba fatty acid reductase (FAR). All three transgenes were under the control of the regulatory sequence of the seed-specific Glycinin promoter. The presence of mRNA of these transformed cDNAs was confirmed by RT-PCR. Gas chromatographic analysis of transgenic seeds indicated that jojoba KCS and FAR generated a substrate pool that contains very long chain fatty acids and long chain primary fatty alcohols. The Arabidopsis transgenic line ectopically expressing the At5g55380 WS produced wax esters in the seed oil.

In this thesis we also presented a reverse genetic approach using T-DNA insertion mutants. Biochemical analysis of plant lipids showed that the At5g55380 mutants did not differ from the wild type, indicating that this gene is redundant in producing wax esters.